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An insoluble DNA polymerase activity that replicates the intact chromatin template at 85% of the rate found in vivo has been partially characterized. HeLa cells, encapsulated in agarose microbeads, are lysed using an isotonic salt concentration: the resulting encapsulated nuclei contain polymerase associated with a nucleoskeleton and the unbroken template. This preparation can be manipulated freely without aggregation or breaking the DNA and yet is accessible to enzymes and other probes. The major activity, which is sensitive to aphidicolin, is found only in S-phase nuclei and replicates DNA semi-conservatively, forming intermediates that are ligated efficiently into larger products.

Type

Journal article

Journal

J Mol Biol

Publication Date

05/11/1986

Volume

192

Pages

65 - 76

Keywords

Autoradiography, Cell Cycle, Chromatin, DNA, DNA Polymerase II, DNA Replication, DNA-Directed DNA Polymerase, HeLa Cells, Humans, Hydrogen-Ion Concentration, Magnesium, Magnesium Chloride, Nucleotides